The present application claims priority to German Application No. DE10030702.7 filed Jun. 23, 2000, the entire contents of which are incorporated herein by reference
1. Field of the Invention
The invention relates to a process for preparing D-pantothenic acid using Coryneform bacteria in which the pfkA gene is enhanced.
2. Discussion of the Background
Pantothenic acid is a vitamin of commercial importance which is used in cosmetics, medicine, human nutrition and animal nutrition.
Pantothenic acid can be prepared by chemical synthesis, or biotechnologically by fermentation of suitable microorganisms in suitable nutrient solutions. In chemical synthetic applications DL-pantolactone is an important intermediate stage. It is prepared in a multi-stage process from formaldehyde, isobutylaldehyde and cyanide. In further process steps, the racemic mixture is separated, D-pantolactone is subjected to a condensation reaction with xcex2-alanine which yields the desired D-pantothenic acid.
The advantage of the fermentative preparation by microorganisms lies in the direct formation of the desired stereoisomeric D-form, which is free from L-pantothenic acid.
Various types of bacteria, such as, Escherichia coli, Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes, and also yeasts, such as, Debaromyces castellii, can produce D-pantothenic acid in a nutrient solution which comprises glucose, DL-pantoic acid and xcex2-alanine, as shown in EP-A 0 493 060. EP-A 0 493 060 further shows that in the case of Escherichia coli, the formation of D-pantothenic acid is improved by amplification of pantothenic acid biosynthesis genes from E. coli which are contained on the plasmids pFV3 and pFV5 in a nutrient solution comprising glucose, DL-pantoic acid and xcex2-alanine.
EP-A 0 590 857 and U.S. Pat. No. 5,518,906 describe mutants derived from Escherichia coli strain IFO3547, such as FV5714, FV525, FV814, FV521, FV221, FV6051 and FV5069, which carry resistances to various antimetabolites, such as salicylic acid, xcex1-ketobutyric acid, xcex1-hydroxyaspartic acid, O-methylthreonine and xcex1-ketoisovaleric acid. These strains produce pantoic acid in a nutrient solution comprising glucose, and produce D-pantothenic acid in a nutrient solution comprising glucose and xcex2-alanine. EP-A 0 590 857 and U.S. Pat. No. 5,518,906 also show that after amplification of the pantothenic acid biosynthesis genes contained on the plasmid pFV31, in the abovementioned strains, the production of D-pantoic acid in nutrient solutions comprising glucose and the production of D-pantothenic acid in a nutrient solution comprising glucose and xcex2-alanine is improved.
The knowledge with respect to processes for preparing D-pantothenic acid with the aid of Corynebacterium glutamicum are known only in some instances in the literature. Sahm and Eggeling (Applied and Environmental Microbiology 65(5), 1973-1979 (1999)) thus report on the influence of over-expression of the panB and panC genes and Dusch et al. (Applied and Environmental Microbiology 65(4), 1530-1539 (1999)) report on the influence of the panD gene on the formation of D-pantothenic acid.
However, there remains a need for improved methods of producing pantothenic acid in Coryneform bacteria. On a commercial or industrial scale even small improvements in the yield of pantothenic acid, or the efficiency of their production, are economically significant. Prior to the present invention, it was not recognized that enhancement of the pfkA gene in Coryneform bacteria would improve pantothenic acid.
One object of the present invention, is providing a new process for producing D-pantothenic acid by culturing a Coryneform bacteria comprising an enhanced pfkA gene and collecting the D-pantothenic acid produced. In preferred embodiments of the invention, the pfkA gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or which pfkA gene copmrprise SEQ ID NO:1. In another embodiment, the pfkA gene comprises those nucleotide sequences which hybridize under stringent conditions to SEQ ID NO:1 and encode a polypeptide having phosphofructokinase activity where the stringent conditions comprise washing in 5xc3x97 SSC at a temperature of from 50 to 68xc2x0 C.
Another object of the present invention is to prepare D-pantothenic acid having the enhance pfkA gene and also having enhanced expression of one or more of panB, panC, and/or ilvD.
In one embodiment the pfkA gene is enhanced by overexpression.
The above objects highlight certain aspects of the invention. Additional objects, aspects and embodiments of the invention are found in the following detailed description of the invention.